Project Summary The human selenoproteome comprises at least 25 selenoproteins. The majority are selenoenzymes that require selenium (Se) in the form of Selenocystein (Sec) for proper enzymatic activity. Some serve as antioxidants or oxido-reductases [glutathione peroxidases (GPx) and thioredoxin reductases], in thyroid hormone metabolism (deiodinases), in Se transport, storage, and delivery to the brain (SePP) and in sperm viability (PHGPx). The machinery for Sec incorporation recodes the UGA codon and requires cis-acting sequences present in the mRNA of all selenoproteins, the in frame UGA and the Sec insertion sequence (SECIS), while Sec-specific tRNASec, and SECIS-binding protein (SECISBP2 or SBP2) are some of the required trans-acting factors. SBP2 is believed to be the major determinant of Sec incorporation as its immunodepletion eliminates Sec incorporation. A not well understood but distinct hierarchy exists in the synthesis of selenoproteins as their expression is differentially affected by Se deficiency, and preferential SECIS recognition by SBP2 was demonstrated. Important medical clues to the consequences of impaired selenoprotein synthesis became apparent with the report of mutations in the SBP2 gene, causing partial SBP2 deficiency in children of several families. Affected subjects presented with transient growth delay and abnormal thyroid function tests resulting from altered thyroid hormone metabolism due to deficiency in the deiodinases. New reports of SBP2 gene mutations describe additional features, a complex phenotype with congenital myopathy and developmental delay in one case, and azoospermia, sensorineural hearing loss in another, thus reflecting multiple selenoprotein deficiencies. Animal models for this new genetic defect are required to answer the many questions raised by the human phenotype. To address this need, I designed a research plan to generate mouse models with Sbp2 deficiency, using recombineering techniques. As redundancy in SBP2 function was not found in-vitro and lack of SBP2 is putatively lethal, I will engineer a Sbp2KI mouse for a C- terminus mutation reported in a patient with a severe phenotype, and an inducible Sbp2KO mouse. The unlimited access to tissues will help distinguish in-vivo the different layers of regulation and selenoprotein hierarchy. Initial investigations of these mice will uncover the underlying mechanisms for the thyroid phenotype, infertility, growth delay, the importance of the putative C-terminal functional domain in-vivo, and the specific cause for the myopathy. The study of the ageing animals will allow close monitoring for manifestations later in life, and other presumed phenotypes, including cancer, neurodegenerative disorders and immune dysfunction. These investigations are relevant to multiple physiological functions and pathways, and will help elucidate the mechanisms underlying selenoprotein-mediated pathology. Ultimately, these animal models will make possible in-vivo testing of various compounds with potential therapeutic properties applicable to humans, thus making this model a necessary tool. PUBLIC HEALTH RELEVANCE: A unique insight into selenoprotein biology was provided by the recent identification of SBP2 gene mutations in humans. The different layers of selenoprotein regulation and hierarchy will be defined by studying the phenotype of an Sbp2 gene knock-in mouse model and that of temporally controlled and tissue specific Sbp2 gene knock-out mouse. These investigations are relevant to multiple physiological functions and pathways, and will help elucidate the mechanisms underlying selenoprotein-mediated pathology